Angiotensin converting enzyme inhibitory peptide

ABSTRACT

To provide ACE inhibitory peptides which can effectively inhibit ACE by a small amount of ingestion and have no fear of causing side effects and which can be orally ingested easily during daily life by persons having high blood pressure, and compositions comprising the peptides. The peptides represented by the following structural formulae (1) to (9), and salts thereof are provided. (1) Asp-Arg-Pro, (2) Asn-Trp, (3) Val-Gly-Leu, (4) Ile-Gly-Val, (5) Gly-Val-Pro, (6) Ile-Pro-Tyr, (7) pyroGlu-Pro, (8) Tyr-Thr, (9) Pro-Trp

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/120,531, filed Mar. 23, 2011, which is a U.S. national stageapplication and claims the benefit of PCT application Serial No.PCT/JP2009/052108, filed Feb. 6, 2009, which claims priority to JapaneseApplication No 2009-008503, filed Jan. 19, 2009, which is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to peptides which exert a function ofreducing blood pressure by inhibiting angiotensin converting enzyme,compositions comprising these peptides and production methods thereof.

BACKGROUND OF THE INVENTION

Angiotensin converting enzyme (to be referred to as ACE hereinafter) isa kind of carboxypeptidase exists in the animal body and producesangiotensin II by digesting one of the peptide bonds of angiotensin I.The angiotensin II is a peptide hormone having a strong vasopressoractivity and causes hypertension through vasoconstriction, accelerationof aldosterone secretion and the like. Accordingly, inhibition of theACE activity makes control of angiotensin II production and treatment ofhypertension possible. Up to this time, medicaments such as captopriland the like have been put into practical use and broadly used as ACEinhibitors. However, since these medicaments have strong actions, thereis a possibility of causing side effects and it is necessary to payattention to side effects such as dry cough and the like in the case ofmany ACE inhibitors.

On the other hand, since the angiotensin I as the substrate of ACE is apeptide, studies have been carried out on the provision of ahypertension treating agent which competitively inhibits ACE by peptidesderived from a natural source and shows no side effects (e.g., seePatent References 1 to 5). Since these peptides have mild actions, highsafety is expected. On the other hand, there is a problem in that apeptide or a composition containing the peptide must be ingested in arelatively large amount in order to ingest its effective amount.Additionally, since many of these peptides have the whole hydrophobicproperty and it is considered that such peptides generally have strongbitterness (e.g., see Patent Reference 6, Non-patent References 1 and2), there is also a problem of having a difficulty in orally taking themin a large amount.

-   Patent Reference 1: JP-A-4-091097-   Patent Reference 2: JP-A-5-262790-   Patent Reference 3: JP-A-6-040944-   Patent Reference 4: JP-A-7-188282-   Patent Reference 5: JP-A-10-175997-   Patent Reference 6: JP-A-2006-75064-   Non-patent Reference 1: Wenyi Wang et al., Comprehensive Reviews in    Food Science and Food Safety, 2005, (4), p. 63-78-   Non-patent Reference 2: “Kaitei Shinpan Shokuhin Kagaku (Revised New    Edition Food Chemistry)” edited by Masao Fujimaki et al., Asakura    Shoten, March, 1976, p. 117-118

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

A problem to be solved by the present invention is to provide ACEinhibitory peptides which effectively inhibit ACE by a small amount ofingestion; have no fear of causing side effects; and show lessbitterness and which can be orally ingested easily during daily life bypersons having high blood pressure, compositions comprising the peptidesand production methods thereof.

Means for Solving the Problems

With the aim of solving the above-mentioned problems, the inventors ofthe present invention have conducted intensive studies and found as aresult peptides which have chemical structures (amino acid sequences) inwhich the presence of an ACE inhibitory action has not so far been knownand have markedly strong action to accomplish the present invention.

Namely, according to the present invention,

[1] Angiotensin converting enzyme inhibitory peptides represented by thefollowing structural formulae (1) to (9):(1) Asp-Arg-Pro, (2) Asn-Trp, (3) Val-Gly-Leu, (4) Ile-Gly-Val, (5)Gly-Val-Pro, (6) Ile-Pro-Tyr, (7) pyroGlu-Pro, (8) Tyr-Thr and (9)Pro-Trp and salts thereof;[2] An angiotensin converting enzyme inhibitor, which comprises at leastone of the peptides described in the above [1];[3] A composition which comprises at least one of the peptides describedin the above [1] and alleviates symptoms of high blood pressure; and[4] A method for producing the peptides described in the above [1],wherein soybean is mixed and stirred at 25° C. or more with a kojifungus culture are provided.

Effect of the Invention

By the present invention, ACE inhibitory peptides which effectivelyinhibit ACE by a small amount of ingestion; have no fear of causing sideeffects; and show less bitterness and which can be orally ingestedeasily during daily life by persons having high blood pressure,compositions comprising these peptides and production methods thereofare provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a figure showing a UV peak when Asp-Arg-Pro was isolated froma composition 1.

FIG. 2 is a figure showing changes in the blood pressure value when thecomposition 1 was administered to high blood pressure rats for aprolonged period of time.

BEST MODE FOR CARRYING OUT THE INVENTION

The following describes the present invention in detail. The peptides ofthe present invention (1) Asp-Arg-Pro, (2) Asn-Trp, (3) Val-Gly-Leu, (4)Ile-Gly-Val, (5) Gly-Val-Pro, (6) Ile-Pro-Tyr, (7) pyroGlu-Pro, (8)Tyr-Thr and (9) Pro-Trp are dipeptides or tripeptides in which eachamino acid residue was expressed by the generally used three letter codeand pyroGlu represents pyroglutamic acid residue. These peptides can beproduced, for example, by a chemically synthesizing method. Asconventionally known methods, for example, there are a liquid phasemethod in which amino group of the amino terminus side amino acid isprotected with a benzyloxycarbonyl group and the carboxyl group isactivated with a p-nitrophenyl ester group and condensed with thecarboxyl terminus side amino acid in the presence of triethylaminefollowed by removal of the protecting group by catalytic reduction orwith trifluoroacetic acid and a solid phase method in which the carboxylterminus side amino acid is bound to a polymeric solid phase support andamino acids after carrying out protection of amino group and activationof carboxyl group are bound thereto one by one by peptide bindingfollowed by removal of the amino acid side chain protecting group bycutting out from the solid phase support using trifluoroacetic acid,hydrogen fluoride or the like. The production can be effected by both ofthe methods. In this connection, similar effects can be expected fromnot only a peptide simple substance but also its salt with aphysiologically acceptable ion.

Additionally, the peptides of the present invention can also be producedusing a column chromatography or the like, by separation andpurification from a composition prepared by hydrolyzing a proteincontaining said amino acid sequence with an appropriate protease agent.In that case, the intended effect can be obtained even when ingested inthe form of a composition containing said peptide. This is moredesirable because mass production is easy and production cost is low, incomparison with the aforementioned chemical synthesis methods.

As the protein source in the case of obtaining the peptide of thepresent invention by hydrolysis of protein, although any material may beused as long as the object of the present invention can be achieved,preferably, it is suitable to use a leguminous plant. More preferably,it is desirable to use soybean from the viewpoints that its cultivationquantity is large; its cost is low; its protein content is large; itseating experience is rich; and taste of the composition after hydrolysisis good. The kind of soybean includes a yellow soybean, a red soybean, ablack soybean and the like and a yellow soybean is particularlydesirable. As the mode of soybean, whole soybean (whole fat soybean),defatted soybean, purified soybean protein and the like can be used assuch or by applying an optional protein denaturation treatment aftercrushing and pulverization. As the protein denaturation treatment, theindustrially broadly used pressure cooking is preferable.

As the protease agent in the case of obtaining the peptide of thepresent invention by hydrolysis of protein, although any material may beused as long as the object of the invention can be achieved, it issuitable to use a koji fungus culture from the viewpoints that itsprotease activity is high; its cost is low; its eating experience isrich; its safety is guaranteed; and taste of the composition afterhydrolysis is good. As the koji fungus, Aspergillus oryzae and/orAspergillus sojae is particularly suitable. These microorganisms havebeen used from ancient times for the protein degradation in fermentationproduction of soy sauce and soybean paste, and their safety has alsobeen mentioned in the list of GRAS (Generally Recognized As Safe) andapproved by Food and Drug Administration (FDA), USA. The koji fungusculture is a product obtained by inoculating and culturing a koji fungususing soybean, wheat, rice etc. as the medium. It is classified into aliquid koji culture and a solid koji culture based on the difference inculturing method and both cases can be used in the present invention.For example, a liquid koji culture can be obtained by inoculating a kojifungus into a liquid medium containing from 1% to 5% of soybean, wheat,wheat bran and the like and culturing it at from 25° C. to 40° C. forfrom 24 hours to 120 hours. Also, a solid koji culture mixture can beobtained by inoculating a koji fungus on a solid medium containingsoybean, wheat and the like and culturing it at from 25° C. to 40° C.for from 24 hours to 120 hours.

As the conditions of the aforementioned hydrolysis reaction, from 10% to70%, preferably from 30% to 50%, in final concentration of a koji fungusculture is mixed with from 5% to 40%, preferably from 15% to 30%, infinal concentration of soybean; from 0% to 25%, preferably from 6% to18%, in final concentration of salt; and further from 0% to 85% in finalconcentration of water, and final concentrations of soybean and salt areadjusted to the aforementioned ranges, followed by the reaction at from25° C. to 60° C., preferably from 30° C. to 55° C., at an agitation rateof from 10 rpm to 150 rpm for from 12 hours to 240 hours, preferablyfrom 24 hours to 168 hours. For the purpose of optionally improvingflavor, it is possible to add a yeast strain belonging to the genusZygosaccharomyces or the like and simultaneously carry out thefermentation.

Despite of containing the peptide of the present invention, thecomposition prepared by the aforementioned production method can bedeliciously ingested as food because bitterness can hardly be felt andit has mellow and rich deliciousness and body and mild and superioraroma, which is and markedly different form the conventionally known ACEinhibitory peptide-containing compositions.

The methods for separating and purifying the peptide of the presentinvention from the aforementioned composition include ultrafiltration,dialysis, various types of chromatography and the like can be mentioned.These are generally broadly used methods. Particularly, a cationexchange chromatography and a reverse phase chromatography are effectivefrom the viewpoint of treating amount of samples and purificationefficiency.

The peptide of the present invention obtained in this manner can be usedby oral or parenteral administration as an ACE inhibitor, namely a bloodpressure increase inhibiting/reducing agent. In accordance with theusual way, it can be made into forms such as tablets, granules, powders,capsules and the like in the case of the oral administration, and can bemade into forms such as injection preparations, percutaneouspreparations, suppositories and the like in the case of parenteraladministration.

Additionally, it can be used in food and drink for the purpose ofpreventing and/or treating hypertension (e.g., a healthy food, ahealth-conscious food, a functional food, a food for specified healthuses and the like). The peptide of the present invention can be producedby adding it to various types of food and drink, and since its taste isgood even in the form of a peptide-containing composition as describedabove, its addition to various types of food and drink is easy.Furthermore, it is also easy to ingest the composition itself. Examplesof the food and drink obtained in this manner include soy sauce, soysauce containing product, powder soy sauce, soybean paste, tsuyu andsoups, broths, soybean milk, fermented milk, soft drink, concentrateddrink stock liquid and adjusting powder, liquors, oil and fat-containingfood, noodles, processed seafood, processed meat food, semi-solid food,pasty food, solid food and the like.

Although the dose of these peptides varies depending on the preventingor treating purpose and methods, within the range of from 0.01 mg to 100mg as peptide per day is desirable. Additionally, in the case ofingesting it as a various types of food and drink, it is desirable thatthe ACE inhibitory activity represented by IC₅₀ becomes 1 mg/ml or lessas a whole.

The following illustratively describes the present invention withreference to examples, the technical scope of the present invention isnot limited to these examples.

Example 1 Production of Peptide by Chemical Synthesis Method

Production of Asn-Trp is exemplified in the following. In thisconnection, as the reagents other than the particularly mentioned ones,those produced by Wako Pure Chemical Industries, Ltd. were used.Firstly, 120 mg of L-tryptophan hydrochloride was dissolved in 2 ml ofdimethylformamide, and 150 μl of triethylamine and 300 mg of Z-Asn-Onp(manufactured by KOKUSAN CHEMICAL Co., Ltd.) were added and stirred atroom temperature for 24 hours. Next, 20 ml of 1% aqueous ammonia wasadded and allowed to stand at −10° C. for 1 hour. The formed precipitatewas collected by filtration and the precipitate was washed with coldwater. The precipitate was dissolved in 20 ml of methanol; 20 mg ofpalladium activated carbon was added thereto; and, after nitrogen gassealing, a catalytic reduction reaction was carried out. The reactionwas carried out at room temperature for 3 days by contacting withhydrogen gas while with under atmospheric pressure. After the reaction,the insoluble matter was removed by filtration and methanol wasevaporated, followed by dissolution in distilled water. Next,fractionation purification was carried out by an ODS column (Cosmosil5C18-ARII 20×250 mm, manufactured by Nakalai Tesque) connected to anHPLC (LC-8A, manufactured by Shimadzu Corp.). By using ultrapurewater+0.1% trifluoroacetic acid (TFA) as the eluent A, andacetonitrile+0.1% TFA as the eluent B, gradient elution was carried outin accordance with the usual way. Each fraction was analyzed by a thinlayer chromatography (TLC), and 87 mg of Asn-Trp was obtained from afraction which showed a spot with a ninhydrin reagent. The chemicalstructure of it was confirmed in accordance with the usual way using aprotein sequencer (Procise 492, manufactured by Applied Biosystems,Inc.), an NMR (ADVANCE 500, manufactured by Bruker) and an LC-MS (1100,manufactured by Agilent Technologies and QSTAR Elite, manufactured byApplied Biosystems, the column was Develosil RPAQUEOUS-AR, manufacturedby NOMURA CHEMICAL CO., LTD.).

ACE inhibitory activity of the peptides obtained by the aforementionedsynthesis method was measured. As the measuring method, it was carriedout by a method in which the method of Yamamoto et al. (Setsuko Yamamotoet al., Journal of the Japan Society of Chest Diseases, 1980, 18, p.297-302) was partially modified. Namely, 140 μl of sample solution, 10μl of enzyme solution (0.3 U/ml of ACE (rabbit lung-derived,manufactured by SIGMA) and 50 mM borate buffer (pH 8.3) were added to100 μl of a substrate solution (12.5 mM Hippuryl-His-Leu (manufacturedby SIGMA), 100 mM borate buffer pH 8.3, 1M NaCl), and the reaction wascarried out at 37° C. for 30 minutes. Subsequently, the reaction wasstopped by adding 250 μl of 1N hydrochloric acid and, after stirring byadding 1.5 ml of ethyl acetate and subsequent centrifugation, 1 ml ofthe ethyl acetate layer was concentrated and dried by centrifugalconcentration followed by dissolution in 1 ml of ultrapure water tomeasure the absorbance at 228 nm. The ACE inhibitory ratio isrepresented by the following formula.

ACE inhibitory ratio(%)={1−(ODs−ODsb)/(ODc−ODcb)}×100

In this connection, ODs is the absorbance when the reaction was carriedout by adding the enzyme solution to a sample as described above; ODsbis the absorbance when ultrapure water was added instead of the enzymesolution; ODc is the absorbance when ultrapure water was added insteadof the sample solution; and ODcb is the absorbance when ultrapure waterwas added instead of the enzyme solution and sample solution.Additionally, the concentration of a sample which gives 50% of theenzyme activity inhibitory ratio on this reaction system is regarded asthe IC₅₀ value.

ACE inhibitory activities of the peptides of the present invention areshown in Table 1. In this connection, the IC₅₀ value of about 400species of the main ACE inhibitory peptides so far reported wasapproximately from 1 μM to 1000 μM (“Shoku no Kagaku Library 3 ShokuhinSeibun no Hataraki (Science Library of Food 3 Action of FoodComponents)”, edited by Koji Yamada, Asakura Shoten, March, 2004, p.63-67). Since the peptides of the present invention have sufficientlystrong activities in comparison with the conventionally known peptides,it is considered that they are industrially useful.

TABLE 1 Amino Acid ACE Inhibitory Activity Sequences (IC₅₀, μM)Asp-Arg-Pro 79 Asn-Trp 25 Val-Gly-Leu 257 Ile-Gly-Val 212 Gly-Val-Pro100 Ile-Pro-Tyr 79 pyroGlu-Pro 106 Tyr-Thr 35 Pro-Trp 186

Example 2 Production of Peptide by a Method in which Soybean is Degradedwith Koji Fungus Culture Mixture

Spores of a koji fungus Aspergillus sojae were added to a solid mediumcontaining equivalent amounts of heat-denatured whole soybean and wheatand cultured at from 25° C. to 40° C. for 72 hours to obtain a kojifungus culture. Next, 14 kg of heat-denatured whole soybean, 2 kg ofkoji fungus culture, 16 liters of water and 3 kg of salt were mixed andstirred at 100 rpm and at 45° C. for 5 days to obtain moromi. Next, ayeast strain: Zygosaccharomyces rouxii was mixed therewith at a ratio of1.5×10⁶ cells/ml and allowed to stand still at 25° C. for 7 days. Next,the insoluble solid matter was removed from the moromi using a hydraulicfilter press to obtain a supernatant. By further carrying out enzymedeactivation and sterilization through heating at 117° C. for 5 secondsusing an HS sterilizer (manufactured by HISAKA WORKS, LTD.) followed bystanding still at 50° C. for 3 days, 20 liters of a supernatant alonecontaining no sediment was collected (composition 1).

By electrodialysis, 5 L of the composition 1 was desalted to about 0%salt and loaded on an ODS column (SP-120-40/60-ODS-B, manufactured byDAISO CO., LTD.) having a column volume of 18 liters. Using distilledwater containing 0.1% TFA as the eluent A, and 30% acetonitrilecontaining 0.1% TFA as the eluent B and acetonitrile containing 0.1% TFAas the eluent C, linear gradient elution was carried out for 20 hoursfrom A to B and then for 5 hours from B to C. By setting the flow rateto 45 ml/min, each 1600 ml fractions were obtained. Six fractionsthereof were combined and concentrated by evaporator, and lyophilized toobtain a freeze-dried powder. The ACE inhibitory activity (IC₅₀ value)thereof was measured.

The above-mentioned freeze-dried powder was dissolved in ultrapure waterand then loaded on an ODS column (Cosmosil 5C18-ARII 20×250 mm,manufactured by Nakalai Tesque) connected to a high performance liquidchromatography (HPLC, manufactured by Shimadzu Corpration). By usingultrapure water containing 0.1% TFA as the eluent A, and 70%acetonitrile containing 0.1% TFA as the eluent B, and after 10 minutesof holding with B: 0%, linear gradient elution was carried out for 70minutes from B: 0% to B: 30% and then for 20 minutes until B: 100%. Bysetting the flow rate to 5 ml/min, each 7.5 ml fractions were obtained.After collecting 100 μl of sample was collected from each fraction,centrifugal concentration were carried out, followed by dissolutionagain in 500 μl of ultrapure water to measure the ACE inhibitoryactivity.

Furthermore, each fraction having the ACE inhibitory activity was loadedon a C30 column (Develosil RPAQUEOUS-AR 20×250 mm, manufactured byNOMURA CHEMICAL CO., LTD.). Using ultrapure water+0.1% TFA as the eluentA, and 40% acetonitrile+0.1% TFA as the eluent B, fractionation wascarried out with a fraction size of 6 ml, at a flow rate of 5 ml/minunder gradient conditions of (A 100%:10 minutes)→(gradient until B30%:60 minutes)→(gradient until B 100%:30 minutes). Its chromatogram isshown in FIG. 1. When the ACE inhibitory activity was measured, theactivity was found only on the peak shown by arrow. When the fractionwas concentrated by an evaporator and structural analysis using aprotein sequencer, an NMR and an LC-MS was carried out, it wasAsp-Arg-Pro. In the same manner, Asn-Trp, Val-Gly-Leu, Ile-Gly-Val,Gly-Val-Pro, Ile-Pro-Tyr and pyroGlu-Pro were isolated from otherfractions.

Using the aforementioned LC-MS, the peptides of the present inventioncontained in the composition 1 were determined. Firstly, a calibrationcurve was prepared using a peptide obtained by a chemical synthesis asthe standard. Next, the composition 1 was desalted by electrodialysisand optionally diluted to carry out its analysis to calculate thecontent from the detected amount of ion derived from each peptide. Theresults are shown in Table 2.

TABLE 2 Amino Acid Content in Composition 1 Sequences (μg, ml)Asp-Arg-Pro 154 Asn-Trp 2 Val-Gly-Leu 1 Ile-Gly-Val 2 Gly-Val-Pro 56pyroGlu-Pro 60 Tyr-Thr 10 Pro-Trp 127

In the same manner, content of the peptides of the present inventioncontained in compositions obtained by production methods havingdifferent conditions are shown in Table 3. As comparative examples,wheat was used instead of soybean, and a protease agent for food use(Alcalase 2.4L-FG, manufactured by NOVOZYMES) instead of the koji fungusculture. From the results of Table 3, it can be understood that soybeanis superior as the material; koji fungus culture is superior as theenzyme preparation; and 25° C. or more is superior as the reactiontemperature. Namely, the peptides of the present invention can beproduced particularly efficiently, by mixing and stirring soybean at 25°C. or more with the koji fungus culture.

TABLE 3 Raw Enzyme Reaction Asp-Arg-Pro Gly-Val-Pro Tyr-Thr Pro-TrpMaterial Preparation Temperature (μg/ml) (μg/ml) (μg/ml) (μg/ml) RemarksSoybean koji fungus 20° C. 33 21 1 90 Present culture Invention 1Soybean koji fungus 25° C. 47 26 3 181 Present culture Invention 2Soybean koji fungus 30° C. 80 37 4 185 Present culture Invention 3Soybean koji fungus 35° C. 95 42 4 161 Present culture Invention 4Soybean koji fungus 50° C. 154 56 10 127 Present culture Invention 5Soybean Alcalase 35° C. 3 n. d. 1 79 Comparative Example 1 Wheat kojifungus 35° C. 3 n. d. 1 7 Comparative culture Example 2

Example 3 Inspection of Hypotensive Action by Long-Term Administrationof Mixed Feed to Rats

After dividing 35 male salt-sensitive hypertensive rats (Dahl-S) of 5weeks of age into 5 groups, each of which consisting of 7 animals, along-term mixed feed administration test was carried out for 30 days.The salt concentration of samples was analyzed by a flame analysis andadjusted by adding salt such that the salt concentration in the feedbecame the same among respective groups. During the testing period,although the feed was ingested freely, the ingested amount was almostthe same among respective groups.

First group (control) (▴ in the drawing): a feed prepared by mixing 25%v/w of a low salt soy sauce (salt concentration 7% w/v) based on thefeed (salt concentration in the feed: 3% w/w).Second group (composition 1) ( in the drawing): a feed prepared bymixing 20% v/v of the aforementioned composition 1 with the low salt soysauce and mixing 25% v/w thereof based on the feed (salt concentrationin the feed: 3% w/w and composition 1 concentration: 1.5% w/w).Third group (a fraction obtained by removing peptides from thecomposition 1) (◯ in the drawing): a feed prepared by mixing theaforementioned ODS-A fraction (a fraction hardly containing peptides,prepared by fractionating the composition 1 by an ODS column) with thelow salt soy sauce and mixing 25% v/w thereof based on the feed (saltconcentration in the feed: 3% w/w and ODS-A fraction concentration: 1.3%w/w).Fourth group (a peptide fraction of the composition 1) (□ in thedrawing): a feed prepared by mixing the aforementioned ODS-B fractionand ODS-C fraction (a fraction rich in peptides, prepared byfractionating the composition 1 by an ODS column) with the low salt soysauce in response to the weight ratio and mixing 25% v/w thereof basedon the feed (salt concentration in the feed: 3% w/w and totalconcentration of ODS-B and ODS-C fractions: 0.2% w/w).Fifth group (a peptide fraction degraded by hydrochloric acid) (▪ in thedrawing): a feed prepared by completely degrading the ODS-B fraction andODS-C fraction by hydrochloric acid degradation in accordance with theusual way and mixing the resulting peptides in the same manner as in thefourth group (salt concentration in the feed: 3% w/w and totalconcentration of ODS-B and ODS-C fractions: 0.2% w/w).

Using a noninvasive sphygmomanometer MK-2000, measurement of systolicblood pressure was carried out at an interval of one week from the timeof commencement of the test. Results of these are shown in FIG. 2. Inthis connection, statistical treatment was carried out by Tukey'smultiple comparison test using the systolic blood pressure value on the30^(th) day after the administration.

From the results of FIG. 2, the composition 1 containing the peptides ofthe present invention significantly inhibited increase of bloodpressure. Additionally, while hypotensive action was found in thepeptide-containing fractions, the hypotensive action was lost by thedegradation of peptides. Therefore, it was shown that the hypotensiveaction of composition 1 is derived from peptides.

Example 4 Sensory Evaluation

The sensory evaluation was carried out using the composition 1 andComparative Example 1 described in Example 2. By a panel of 8 members,“strength of bitterness” and “desirableness of taste” were evaluated bya paired comparison method. From the results of Table 4, it can beunderstood that although the fermented seasoning of the presentinvention is rich in peptides, it shows less bitterness and has asuitable taste. Although a large number of peptide production methodswhich use protease agents have so far been disclosed, it is consideredthat the present invention is superior to the related art in terms ofits taste.

TABLE 4 Sample which shows strong Sample which has bitterness preferabletaste Composition 1 1 8 Comparative 7 0 Example 1

While the present invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to oneskilled in the art that various changes and modifications can be madetherein without departing from the spirit and scope of the presentinvention.

This application is based on a Japanese patent application filed on Jan.19, 2009 (Japanese Patent Application No. 2009-008503), the entirecontents thereof being thereby incorporated by reference. Additionally,all of the references cited herein are incorporated as a whole.

INDUSTRIAL APPLICABILITY

The angiotensin converting enzyme inhibitory peptides obtained by thepresent invention effectively inhibit ACE by a small amount of ingestionand have no fear of causing side effects and can be orally ingestedeasily during daily life by persons having high blood pressure.Additionally, by the presentation of the production methods ofcompositions which contain said peptides; are excellent in taste; havehigh safety; and can be easily ingested as food, it becomes possible togreatly contribute to the improvement of quality of life of personshaving high blood pressure.

1. An angiotensin converting enzyme inhibitory peptide represented by the following structural formula: pyroGlu-Pro and salts thereof.
 2. An angiotensin converting enzyme inhibitor, which comprises the peptide described in claim
 1. 3. A composition which comprises the peptide described in claim 1 and alleviates symptoms of high blood pressure.
 4. A method for producing the peptide described in claim 1, wherein soybean is mixed and stirred at 25° C. or more with a koji fungus culture. 